AUTHOR=Wolf Natalie , Bussmann Michael , Koch-Koerfges Abigail , Katcharava Nino , Schulte Julia , Polen Tino , Hartl Johannes , Vorholt Julia A. , Baumgart Meike , Bott Michael TITLE=Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of Corynebacterium glutamicum JOURNAL=Frontiers in Microbiology VOLUME=11 YEAR=2020 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.00087 DOI=10.3389/fmicb.2020.00087 ISSN=1664-302X ABSTRACT=

In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ΔcyaB mutant. While no growth defect of the ΔcyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the ΔcyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown ΔcyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F1FO-ATP synthase and numerous transport processes. The impaired membrane potential of the ΔcyaB mutant could be due to a decreased expression of the cytochrome bc1-aa3 supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the ΔcyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original ΔcyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the ΔcyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum.